Electrostatic effects in proteins govern many essential biological processes, including enzymatic catalysis, bioenergetics, and all other processes that involve H+ transport and e- transfer. To understand the structural basis of function in the proteins that perform these essential biochemical reactions, it is necessary to understand the relationship between structure and the electrostatic properties of proteins. The specific aims of this proposal describe experimental studies to examine fundamental aspects of protein electrostatics. The studies are focused on the properties of internal ionizable groups because those are the ones that are essential for function and those are the ones whose properties are not understood. Charges are not as compatible with the hydrophobic environment in the protein interior as they are in water. For this reason the properties of internal groups are unusual; their pKa values are highly anomalous, shifted in the direction that promotes the neutral state (elevated pKa values for acidic residues and depressed ones for basic residues). The longterm goal of this project is to understand the molecular factors that determine the pKa values of internal ionizable groups in proteins. This requires understanding all the factors that stabilize charge inside a protein, and the effects of charge on the structure and dynamics of proteins. These studies are only possible because we have developed previously a library of 100 variants of staphylococcal nuclease with internal Lys, Asp, Glu and Arg at 25 internal locations. We have already measured the pKa values of these groups. These pKa values were already used to expose deep flaws in computational models for structure-based calculation of electrostatic effects in proteins. X-ray crystallography, NMR spectroscopy and equilibrium thermodynamics will now be used to determine how the structure and dynamics of proteins are affected by the ionization of internal groups. The properties of internal ion pairs will be studied. Contributions from interactions between charges and permanent dipoles and internal water molecules on the pKa values of these internal groups will be measured. These experiments examine fundamental aspects of protein electrostatics that have never been studied and which could not have been studied until this family of proteins was engineered. The results of these experiments are necessary to describe dielectric relaxation in proteins. They will be used for blind challenges to raise awareness of fundamental flaws with methods for structure-based energy calculations. These experiments will contribute the physical insight necessary to guide the development of new computational methods, and contribute the data needed for stringent benchmarking of existing methods.